EP0613379A1

EP0613379A1 - Therapeutic compounds

Info

Publication number: EP0613379A1
Application number: EP92922888A
Authority: EP
Inventors: Agamemnon Antoniou ICRF Dept of Clinic EPENETOS
Original assignee: Imperial Cancer Research Technology Ltd
Current assignee: Cancer Research Horizons Ltd
Priority date: 1991-11-12
Filing date: 1992-11-10
Publication date: 1994-09-07
Anticipated expiration: 2012-11-10
Also published as: ES2125275T3; EP0613379B1; GB9409156D0; GB2275928B; JPH07504885A; WO1993009813A1; DE69226646T2; GB9123947D0; ATE169504T1; DE69226646D1; GB2275928A; DK0613379T3

Abstract

L'invention concerne un composé comportant un élément destiné à diriger ce composé vers un acide nucléique dans une cellule et une fraction radioactive pouvant détruire la matière biologique contiguë. Il est souhaitable que l'élément dirigeant le composé comprenne un oligonucléotide non codant. L'oligonucléotide non codant peut être complémentaire à l'ADN ou l'ARN faisant spécifiquement partie d'un gène destiné à la protéine mutante ras, la protéine mutante p53, la protéine bcr ou ab1 ou une protéine du VIH (virus immunodéficitaire humain), par exemple les produits géniques du VIH gag, pol, env ou sor. Le composé peut comporter également une partie ciblante afin de cibler le composé vers un type de cellule désiré, par exemple il peut comporter un anticorps monoclonal ou une partie spécifique de celui-ci destinée à un antigène spécifique à une tumeur ou spécifique à un virus.The invention relates to a compound comprising an element intended to direct this compound towards a nucleic acid in a cell and a radioactive fraction capable of destroying the contiguous biological material. It is desirable that the directing element the compound comprise a non-coding oligonucleotide. The non-coding oligonucleotide may be complementary to DNA or RNA that is specifically part of a gene intended for the ras mutant protein, the p53 mutant protein, the bcr or ab1 protein or an HIV protein (human immunodeficiency virus) , for example HIV gene products gag, pol, env or sor. The compound may also have a targeting portion to target the compound to a desired cell type, for example it may include a monoclonal antibody or a specific portion thereof for a tumor specific or virus specific antigen.

Description

THERAPEUTIC COMPOUNDS

The present invention relates to therapeutic compounds useful in the treatment of neoplasms (ie cancers, tumours), viral diseases and other conditions.

It is known, at least on a theoretical basis, to administer antisense oligonucleotides (DNA or RNA) to inhibit the function in vivo of the corresponding sense nucleotides (DNA or RNA). Such antisense oligonucleotides are disclosed by Tullis in EP 92574 Bl .

We now provide improved compounds incorporating such oligonucleotides.

The invention provides a compound comprising a means to direct the compound to a nucleic acid within a cell and a radioactive moiety capable of destroying adjacent biological matter, particularly nucleic acid.

It is preferred that the means to direct the compound comprises an antisense oligonucleotide.

Antisense oligonucleotides are single-stranded nucleic acid, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed. These nucleic acids are often termed "antisense" because they are complementary to the sense or coding strand of the gene. Recently, formation of a triple helix has proven possible where the oligonucleotide is bound to a DNA duplex. It was found that oligonucleotides could recognise sequences in the major groove of the DNA double helix. A triple helix was formed thereby. This suggests that it is possible to synthesise sequence-specific molecules which specifically bind double-stranded DNA via recognition of major groove hydrogen binding sites. By binding to the target nucleic acid, the above oligonucleotides can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking the transcription, processing, poly(A) addition, replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradations.

The antisense oligonucleotide can be used to selectively suppress certain cellular functions. For example, in oncogenic transformed cells, oligonucleotides complementary to the oncogene suppress its expression. An antisense oligonucleotide has been shown to inhibit c-myc protein expression in a human promyelocytic leukaemia cell line, HL60, which over expresses the c-myc proto-oncogene. The antisense oligonucleotide used was complementary to regions of the c-myc mRNA.

Antisense oligonucleotides can also be used to inhibit replication and expression of nucleic acid foreign to the host cells. Antisense oligonucleotides are prepared in the laboratory and then introduced into cells, for example by microinjection or uptake from the cell culture medium into the cells, or they are expressed in cells after transfection with plasmids or retroviruses carrying an antisense gene. Antisense oligonucleotides were first discovered to inhibit viral replication or expression in cell culture for Rous sarcoma virus, vesicular stomatitis virus, herpes simplex virus type 1, simian virus and influenza virus.

Since then, inhibition of mRNA translation by antisense oligonucleotides has

^" been studied extensively in cell-free systems including rabbit reticulocyte lysates and wheat germ extracts. Inhibition of viral function by antisense oligonucleotides has been demonstrated in vitro using oligonucleotides which were complementary to the AIDS HIV retrovirus RNA (Goodchild, J. 1988 "Inhibition of Human Immunodeficiency Virus Replication by Antisense Olϊgodeoxynucleotides", Proc. Natl. Acad. Sci. (USA) 85(15), 5507-11). The Goodchild study showed that oligonucleotides that were most effective were complementary to the poly(A) signal; also effective were those targeted at the 5' end of the RNA, particularly the cap and 5' untranslated region, next to the primer binding site and at the primer binding site. The cap, 5' untranslated region, and poly(A) signal lie within the sequence repeated at the ends of retrovirus RNA (R region) and the oligonucleotides complementary to these may bind twice to the RNA.

The antisense oligonucleotide may be any useful antisense oligonucleotide, for example an oligonucleotide complementary to DNA or RNA specifically forming part of a gene for the mutant ras protein, the mutant p53 protein, the BCR-ABL fused mRNA characteristic of the Philadelphia chromosome in chronic myeloid leukaemia (CML) and acute lymphocytic leukaemia (ALL), or an HIV (human immunodeficiency virus) protein, for example the HIV gag, pol, env or sor gene products. By "specifically", we mean that the complementary DNA or RNA does not normally appear in a normal (non- tumour or non-virally-infected) cell. Further targets for the antisense DNA/RNA include the HIV tRNA (Lys) primer binding site, mRNA splice donor or acceptor sites, the poly A region and the initiator codons of the HIV genes mentioned above.

Thus, in the case of CML, the oligonucleotide,

GCTGAAGGGCTT'TTGAACTCTGCTTA hybridises to BCR exon 3/ABL and exon II junction sequences; and oligonucleotide

^• GCTGAAGGGCTT'CTTCCTTATTGATG hybridises to BCR exon 2- ABL and exon II fusions. In the case of ALL, oligonucleotide

GCTGAAGGGCTT'CTGCGTCTCCAT hybridises to the junction of BCR/ABL. In the above sequences, the circumflex denotes the junction between BCR and ABL exons.

Alternatively, the following sequences may be used:- CTGGTCTAACCAGAGAGAC C (designated BB01 ) ; and GCAAGCTTTATTGAGGCTTA (designated BB02). A control could have the following sequence: CAGTCAGTCAGTCAGTCAGT (designated BB03). BB01 is complementary to the cap or initiator codon of HIV and BB02 is complementary to the poly(A) signal of the HIV genomic RNA. The cap and poly(A) signal lie within the sequence repeated at the ends of the HIV RNA (R region). BB03 is a 20-mer not complementary to the HIV RNA. BB03 has been tested and shown to be inactive (Goodchild, J. supra).

Further HIV oligonucleotides include: CTGCTAGAGATddT;

TGCTAGAGATTTTCCACAC; TTCAAGTCCCTGTTCGGGCGCCAAA; GCGTACTCACCA GTC GCCGC ; CTGCTA GAGATTA A ; ACACCCAATTCTGAAAATGG; and equivalents thereof. Alternatively, the oligonucleotide can target HTV nucleotide sequences which code for a protease necessary for proper viral assembly. Oligodeoxynucleotides blocked at the 3' end by ddT, the isourea group or other chain terminators may prove to be more effective inhibitors. In general, any highly conserved region of the HIV genome which encodes information necessary for viral replication or gene expression (eg protein synthesis) is a potential target for complementary oligodeoxynucleotides. Further, the oligonucleotide can be complementary to the viral mRNA, one strand of an integrated or unintegrated proviral DNA, a DNA-RNA, or RNA-RNA duplexes.

^• Similarly, the antisense oligonucleotide conjugate can be used to inhibit replication or expression of other viruses, for example, herpes viruses in the treatment of herpes. Additionally, in the case of DNA viruses, the oligonucleotides can be complementary to the genomic DNA.

A further embodiment of the invention provides a nucleic acid, preferably DNA, directed to a nucleic acid within a cell wherein the DNA is capable of homologous recombination with the said nucleic acid (DNA) within a cell. In this embodiment it is preferred that the DNA of the invention contains between 100 nucleotides and 50 000 nucleotides, more preferably between 500 and 10 000, or between 1000 and 5000; it is further preferred that the DNA is double-stranded.

The DNA of this embodiment of the invention may be designed to recombine homologously with specific DNA sequences within the cells to be targeted. In particular, cells containing gross chromosomal modifications which for example alter say between 100 bp and 100 kb sections of a chromosome, or have insertions or deletions of between 100 bp and 100 kb, or are translocations between or within chromosomes provide candidate targets for the DNA.

Thus, a cell containing such chromosome modifications may be targeted. An example of such cells are chronic myeloid leukaemia cells which contain the Philadelphia chromosome (as disclosed above) characterised by a translocation which juxtaposes parts of the bcr and parts of the abl genes. DNA of the invention containing between 1000 and 5000 bp of the bcr gene fused to between 1000 and 5000 bp of the abl gene in such a way that this fusion corresponded to the sequence of the bcr-abl fusion on the Philadelphia chromosome may be useful in treating CML.

The foregoing approach may be used to treat diseases other than cancers and ^■ viral infections, and may be applied to the treatment of sepsis, as described below.

TNF-induced Diseases/Symptoms

Examples of antisense oligonucleotides that can be used for preventing or suppressing TNF-induced diseases, for example sepsis, are those complementary to TNF DNA or TNF RNA. For example, oligonucleotides complementary to the following can be used: sequences around the 5' end of the TNF messenger RNA; sequences at the beginning of and within the mRNA region coding for the transmembrane domain of the TNF protein; and sequences within the coding region of the 17kD molecule. Examples of the specific oligonucleotide sequences complementary to the above mRNA regions are:

5'TCTCCCTCTTAGCTGGTCCTCTGC3 ' ; 5'CATGCTTTCAGTGCTCATGGTGTCCTTTC3 ' ;

5'GATCAGGAAGGAGAAGAGGCTGAGGAACAA3 ' ; 5'CTCAGCTTGAGGGTTTGC3\- and 5'TTCGTCCTCCTC AC AGGGC3 ' .

It will be apparent to those skilled in the art that the oligonucleotide used in the treatment of the above diseases, and other applications, can be either an oligodeoxynucleotide or an oligoribonucleotide. Among other factors, the choice will be dependent on the case of synthesis, the efficacy, and the relative stability and special advantages of the oligonucleotides in a particular system. Further, the oligonucleotides can be complementary to either DNA or RNA. It can also bind to either or both single-stranded or double-stranded nucleic acid. The DNA or RNA can be indigenous (cellular) to the cell in question or it can be foreign nucleic acid found in the host cells. The DNA can be cellular or foreign infectious DNA, eg those of viruses, bacteria, yeast, fungi and other parasites. The RNA can be genomic RNA or messenger RNA, for example retroviral genomic RNA or foreign or cellular mRNA. Where the oligonucleotide is complementary to and bound to the genomic DNA or RNA, it inhibits or prevents the nucleic acid from being replicated. By interfering with or inhibiting the replication of the nucleic acid, the oligonucleotide interferes with or inhibits downstream expression of the DNA or RNA in protein synthesis. Where the oligonucleotide is complementary to the messenger RNA it interferes with or inhibits the mRNA from being expressed in protein synthesis.

Of course, the oligonucleotides may be "modified oligonucleotides".

By "modified oligonucleotides" we mean that they may contain phosphorothioate, methylphosphonate or other phosphoramidite internucleosidic linkages as well as, or instead of the usual phosphodiester linkages. Such internucleosidic linkages are less susceptible to nucleolytic degradation, or may confer on the antisense oligonucleotide other preferred pharmacokinetic properties. A further modification that can be made instead of or in addition to the aforementioned modifications is the addition of a component capable of intercalating into the target nucleic acid, and thus stabilising the resultant (antisense oligonucleotide): (target nucleic acid) hybrid. The intercalating component is preferably acridine.

An enhanced inhibitory effect can be obtained by rendering the antisense oligonucleotide, or other means to direct the compound to a nucleic acid within the cell, radioactive.

The radioactive moiety may comprise phosphorus-32. However, more preferably it is iodine- 125, iodine-131 , indium-I l l , rhenium-186, rhenium-188 or yttrium-90, or any other isotope which emits enough energy to destroy neighbouring cells, organelles or nucleic acid. Preferably, the isotopes and density of radioactive atoms in the compound of the invention are such that a dose of more than 4000 cGy (preferably at least 6000, 8000 or 10000 cGy) is delivered to the cell and its organelles, particularly the nucleus.

The radioactive atom(s) may be incorporated in the compound of the invention in known ways. For example, the first portion may be biosynthesized or may be synthesized by in vitro synthesis using in each case suitable radioactive nucleotides, nucleosides or bases, for example. A pre-formed oligonucleotide may be labelled with ^32p using T4 polynucleotide kinase and γ-[^32p]ATP.

EDTA or another chelating agent may be attached to a 5'-phosphate group (FEBS Letters (1984), 172, 43-46) and used to attach ^luIn or ⁹⁰Y, for example. Tyrosine can be esterified to the 3'-hydroxyl group and labelled with ¹²⁵I or ¹³¹L

The compound may additionally comprise a portion capable of targeting the compound to cells generally or to a desired cell type.

By "capable oP we mean capable of targeting the compound as said when the said targeting portion is part of the compound of the invention.

The targeting portion may specifically bind to a cell-type-specific entity or may be specifically taken up by the specific cell type which is the intended target.

The entity recognised may be characteristic of cells in general, so that the antisense oligonucleotide is simply taken up into cells and is therefore exposed less to extracellular nucleases, for example. The specificity of the compound is thus derived solely from the antisense oligonucleotide.

Alternatively, the entity which is recognised may be a suitable entity which is specifically expressed by tumour cells, virally-infected cells, pathogenic microorganisms, cells introduced as part of gene therapy or even specific normal cells of the body into which, for whatever reason, one wishes to introduce the antisense oligonucleotide, but which entity is not expressed, or at least not with such frequency, in cells into which one does not wish to introduce the oligonucleotide. The entity which is recognised will often be an antigen. Examples of antigens include those listed in Table 1 below. A non¬ specific antigen is the transferrin receptor, to which antibodies may be raised, as taught in EP 226 419. Monoclonal antibodies which will bind specifically to many of these antigens are already known (for example those given in the Table) but in any case, with today's techniques in relation to monoclonal antibody technology, antibodies can be prepared to most antigens. The antigen- specific portion may be an entire antibody (usually, for convenience and specificity, a monoclonal antibody), a part or parts thereof (for example an F_ab fragment, F(ab')₂, dab or "minimum recognition unit") or a synthetic antibody or part thereof. A compound comprising only part of an antibody may be advantageous by virtue of being less likely to undergo non-specific binding due to the F_c part. Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H. Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications", J.G.R. Hurrell (CRC Press, 1982). All references mentioned in this specification are incorporated herein by reference. Bispecific antibodies may be prepared by cell fusion, by reassociation of monovalent fragments or by chemical cross-linking of whole antibodies, with one part of the resulting bispecific antibody being directed to the cell-specific antigen and the other to the oligonucleotide. The bispecific antibody can be administered bound to the oligonucleotide or it can be administered first, followed by the oligonucleotide. The former is preferred.

^• Methods for preparing bispecific antibodies are disclosed in Corvalan et al (1987) Cancer Immunol. Immunother. 24, 127-132 and 133-137 and 138-143. Bispecific antibodies, chimaeric antibodies and single chain antibodies are discussed generally by Williams in Tibtech, February 1988, Vol. 6, 36-42, Neuberger et al (8th International Biotechnology Symposium, 1988, Part 2, 792-799) and Tan and Morrison (Adv. Drug Delivery Reviews 2, (1988), 129- 142). Suitably prepared non-human antibodies can be "humanized" in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies. IgG class antibodies are preferred.

Table 1

1. Tumour Associated Antigens

Antigen Antibody Existing Uses

10 Carcino-embryonic {C46 (Amersham) Imaging & Therapy of colon/rectum tumours. Antigen {85A12 (Unipath)

Placental Alkaline H17E2 (ICRF, Travers Imaging & Therapy of testicular and ovarian Phosphatase & Bodmer cancers.

15

Pan carcinoma NR-LU-10 (NeoRx Imaging & Therapy of various carcinomas Corporation) incl. small cell lung cancer.

Polymorphic Epithelial HMFG1 (Taylor- Imaging & Therapy of ovarian cancer,

Mucin (Human milk fat Papadimitriou, ICRF) pleural effusions. globule)

/3-human Chorionic W14 Targeting of enzyme (CPG2) to human 5 Gonadotropin xenograft choriocarcinoma in nude mice. (Searle et al (1981) Br. J. Cancer 44, 137-144).

a Carbohydrate on L6 (IgG2a)> Targeting of alkaline phosphatase. 10 Human Carcinomas (Senter et al (1988) P.N.A. S. 85, 4842- 4846.

CD20 Antigen on B 1 F5 (ϊgG2a)² Targeting of alkaline phosphatase.

Lymphoma (normal and (Senter et al (1988) P.N.A. S. 85, 4842- 15 and neoplastic) 4846.

' Hellstrδm et al (1986) Cancer Res. 46, 3917-3923 ²Clarke et al (1985) P.N.A. S. 82, 1766- 1770

Other antigens include alphafoetoprotein, Ca-125 and prostate specific antigen.

2. Immune Cell Antigens

5 Pan T Lymphocyte OKT-3 (Ortho) As anti-rejection therapy for kidney Surface Antigen (CD3) transplants.

B-lymphocyte Surface RFB4 (Janossy, Royal Immunotoxin therapy of B cell lymphoma. Antigen (CD22) Free Hospital)

10

Pan T lymphocyte H65 (Bodmer, Knowles Immunotoxin treatment of Acute Graft Surface Antigen (CD5) ICRF, Licensed to Xoma versus Host disease, Rheumatoid Arthritis. Corp., USA)

15 3. Infectious Agent-Related Antigens

Mumps virus-related Anti-mumps polyclonal Antibody conjugated to Diphtheria toxin for antibody treatment of mumps.

Hepatitis B Surface Anti HBs Ag Immunotoxin against Hepatoma.

Antigen

If applied to the treatment of CML or ALL, the ligand binding molecules can be monoclonal antibodies against leukaemi

5 associated antigens. Examples of these are: anti-CALLA (common acute lymphoblastic leukaemia-associated antigen), J

BA-3, RFB-1 , BA-2, SJ-9A4 Du-ALL-1 , anti-3-3, anti-3-40, SN1 and CALL2, described in Foon, K.A. et al 1986 Bloo

68(1), 1-31 , "Review: Immunologic Classification of Leukemia and Lymphoma" . The ligand binding molecules can als

be antibodies that identify myeloid cell surface antigens, or antibodies that are reactive with B or T lymphocytes, respectivel

Examples of such antibodies are those which identify human myeloid cell surface antigens or those which are reactive wit

10 human B or T lymphocytes as described in Foon, K.A. Id. Additional examples are antibodies B43, CD22 and CD19 whic

are reactive with B lymphocytes can also be used.

Alternatively, the entity which is recognised may or may not be antigenic but can be recognised and selectively bound to

some other way. For example, it may be a characteristic cell surface receptor such as the receptor for melanocyte-stimulati

15 hormone (MSH) which is expressed in high numbers in melanoma cells. The cell-specific portion may then be a compou

or part thereof which specifically binds to the entity in a non-immune sense, for

example as a substrate or analogue thereof for a cell-surface enzyme or as a

messenger. In the case of melanoma cells, the cell-specific portion may be

MSH itself or a part thereof which binds to the MSH receptor. Such MSH

peptides are disclosed in, for example, Al-Obeidi et al (1980) /. Med. Chem.

32, 174. The specificity may be indirect: a first cell-specific antibody may be

administered, followed by a compound of the invention directed against the first

antibody. Preferably, the entity which is recognised is not secreted to any

relevant extent into body fluids, since otherwise the requisite specificity may

not be achieved.

The targeting portion of the compound of this embodiment of the invention may

be linked to the remainder of the compound by any of the conventional ways

of linking compounds, for example by disulphide, amide or thioether bonds,

such as those generally described in Goodchild, supra or in Connolly (1985)

Nucl. Acids Res. 13(12), 4485-4502 or in PCT/US85/03312. A thiol group can

be introduced at the 5'-end of an aminofunctionalised oligonucleotide (ref:

Nucleic Acids Res. (1991) 19, 4561). This group can be used to attach the

oligonucleotide to a protein, such as a monoclonal antibody or growth factor,

using standard heterobiofunctional protein cross-linking reagents such as m-

maleimϊdobenzoylN-hydroxysuccinimide ester (MBS). These reagents usually

link between a thiol group in one protein and the terminal amino group in a lysine residue in the other protein. Preferably, the linkage is cleavable in

lysosomes by lysosomal enzymes or by the acidic environment to liberate the

antisense oligonucleotide.

The compounds of the invention may be administered in any suitable way,

usually parenterally, for example intravenously, intraperitoneally or, preferably

(for bladder cancers), intra-vesically (ie into the bladder), in standard sterile,

non-pyrogenic formulations of diluents and carriers, for example isotonic saline

(when administered intravenously). If needed, because the compound of the

invention may be immunogenic, cyclosporin or some other immunosuppressant

can be administered to provide a longer period for treatment but usually this

will not be necessary.

Particular tumours suitable for treatment in accordance with either aspect of the

invention include cancers of the uterine cervix, head, neck, brain gliomas,

breast, colon, oesophagus, stomach, liver, pancreas and metastatic forms of any

of these.

If desired, the antisense oligonucleotide can be conjugated with hydrophobic

derivatives as taught in FR 2 649 321 to protect it from nucleases and to

improve transport across cell membranes. The hydrophobic moiety may be

cholesterol as taught by Zon in "Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression", pp 234-247, J.S. Cohen (Ed), CRC Press, Boca Raton,

FL, 1989.

Conjugation of the oligonucleotides to poly-L-lysine may also enhance delivery

of the said oligonucleotides to the cell as disclosed by Stevenson and Iversen

(1989) /. Gen. Virol. 70, 2673-2682, and by LeMaitre et al (1987) Proc. Natl.

Acad. Sci. USA 84, 648-652.

In a similar fashion polyamines conjugated to phosphorothioate oligonucleotides

enhance their cellular uptake as taught in US 5 138 045.

The invention will now be described in detail by reference to the following

Examples and Figures in which Figure 1 shows the effect of non-radioactive

random, c-er&-B2 sense and c-erb-B2 antisense oligonucleotides on the viability

of SKBR3 cells and Figure 2 shows the effect of ¹²⁵I-labelled c-erb-B2 antisense

and c-erb-B2 sense oligonucleotides.

EXAMPLE 1

The chosen oligonucleotide (see below) is labelled at the 3' end by esterifying

thereto a tyrosine group and labelling it with ¹²⁵I by known methods. In order to form a conjugate, oligonucleotides with a reactive sulphydryl group

were synthesised. In the preferred embodiment, the oligonucleotides with

reactive sulphydryl groups were designated BB04, BB05, and BB06, each with

a sequence corresponding to BB01 , BB02, and BB03 respectively (see above).

The following reaction conditions were used:

BB01 20-MER

CTGGTCTAACCAGAGAGACC

MW AMMONIUM SALT 6417.65

MOLAR EXTINCTION AT 260nm 195800

MICROGRAMS PER OD260nm 32.78

PICOMOLES PER OD260nm 5107.25

BASE COMPOSITION: ACGT 6653

Td (blot) 0.1 M Na+ 62

Tm @ 0.1M Na+ , .000001M Probe 58

BB02 20-MER

GCAAGCTTTATTGAGGCTTA

MW AMMONIUM SALT 6453.67

MOLAR EXTINCTION AT 260nm 193700

MICROGRAMS PER OD260nm 33.32

PICOMOLES PER OD260nm 5162.62 BASE COMPOSITION: ACGT 5357

Td (blot) 0.1M Na+ 56

Tm @ 0.1M Na+, .000001M Probe 60

BB03 20-MER

CAGTCAGTCAGTCAGTCAGT

MW AMMONIUM SALT 6423.65

MOLAR EXTINCTION AT 260nm 196900

MICROGRAMS PER OD260nm 32.62

PICOMOLES PER OD260nm 5078.72

BASE COMPOSITION: ACGT 5555

Td (blot) 0.1M Na+ 60

Tm @ 0.1M Na+, .000001M Probe 55

The sulphydryl analogues were as follows:

BB04 21-MER

XCTGGTCTAACCAGAGAGACC

MW AMMONIUM SALT 6417.65

MOLAR EXTINCTION AT 260nm 220400

MICROGRAMS PER OD260nm 29.12

PICOMOLES PER OD260nm 4537.21 BASE COMPOSITION: ACGT 6653

MIXED BASES: YRNMKSWHBVDXZ OOOOGOOOOOOIO

X=ANTITRANSFERRIN RECEPTOR ANTIBODY,

Td (blot) 0.1M Na+ 63

Tm @ 0.1M Na+ , .000001M Probe 58

BB05 21-MER

XGCAAGCTTTATTGAGGCTTA

MW AMMONIUM SALT 6453.67

MOLAR EXTINCTION AT 260nm 217600

MICROGRAMS PER OD260nm 29.66

PICOMOLES PER OD260nm 4595.59

BASE COMPOSITION: ACGT 5357

MIXED BASES: YRNMKSWHBVDXZ 0000000000010

X=ANTITRANSFERRIN RECEPTOR ANTIBODY,

Td (blot) 0.1M Na+ 57

Tm @ 0.1M Na+ , .000001 M Probe 60

BB06 21-MER

XCAGTCAGTCAGTCAGTCAGT

MW AMMONIUM SALT 6423.65

MOLAR EXTINCTION AT 260nm 215000 20

MICROGRAMS PER OD260nm 29.88

PICOMOLES PER OD260nm 4651.16

BASE COMPOSITION: ACGT 5555

MIXED BASES: YRNMKSWHBVDXZ 0000000000010

X=ANTITRANSFERRIN RECEPTOR ANTIBODY,

Td (blot) 0.1M Na+ 61

Tm @ 0.1M Na+, .000001M Probe 55

The oligonucleotides had the following generalised structure (the sulphydryl

group was denoted as X above:

O

II

HS O — O-P-O-5 ' DNA o ιι-

At the same time, a sulphydryl group was added to an anti-HIV antigen

antibody. The oligonucleotide was covalently linked to the MAb through a

disulphide exchange reaction between the sulphydryl groups of the two

compounds. This reaction is described below. Addition of Sulphydryl Group on the Antibody

The addition of a thiol group or an activated disulphide group to an

immunoglobulin is known in relation to the synthesis of immunotoxins (US

Patent No 4,340,535). The procedures disclosed therein regarding addition of

the thiol group to the antibody are incorporated hereby.

Ellman's reagent is added to a solution of antibody in 40 mM phosphate buffer

containing 1 mM EDTA. The final concentration of MAb is 3.2 mg/ml, and

that of Ellman's reagent is 1 mM. Final pH of the mixture is 8. The reaction

is allowed to proceed for 30 minutes at room temperature. At the end of 30

minutes, the reaction mixture is cooled in an ice bucket to 4°C and a ten fold

excess of 2-iminothiolane reagent is added. The reaction mixture is allowed to

continue at 4°C overnight. At the end of the reaction, the excess reagents are

separated on a 1.5 x 15 cm column of Sephadex G-25 equilibrated with 40 mM

sodium phosphate, pH 7.6, containing 0.2 M NaCl and 1 mM EDTA.

Disulphide Linkage of the Oligonucleotide with the Antibody

The derivatised antibody is then covalently linked, in a disulphide exchange,

to the sulphydryl group on the oligonucleotide. This linkage was achieved by

incubating the two components (MAb-IT-TNB at 4 nM and oligonucleotide at 100 nM, final concentration) overnight at 4°C. The sample will turn yellow,

indicating that the TNB group is being released and the desired product is being

formed.

The sample is then passed through a 2.5 x 24 cm column of Sephadex G-25

resin equilibrated with 40 mM sodium phosphate, pH 7.6, containing 0.2 M

NaCl. The separation profile of a standard mixture of proteins may be

compared to MAb.

EXAMPLE 2

Synthesis of Oligonucleotide Antibody Conjugates Through Thioether Bonding

In general, the procedure for forming the oligonucleotide antibody conjugate

consists of reacting antibody, either polyclonal or monoclonal, having a free

amino group with a maleimide-active ester in a suitably buffered solution.

Preferably, the maleimide-active ester is present in about a two-fold molar

excess over antibody, and the pH of the solution is slightly alkaline to maintain

the antibody's amino group in an unprotonated state. The reaction of antibody

with the thioether crosslinker can be followed by monitoring the absorbance of

the solution at a wavelength of about 405 nm. An increase in absorbance at

this wavelength is the result of the dianion leaving group, HNSA, and the reaction of antibody amines to form stable amide bonds. Because hydrolysis

of the crosslinker' s active ester is slow relative to aminolysis, most of the

leaving groups' absorbance is due to amide bond formation. The reaction of

antibody with the crosslinker is for a time sufficient to introduce about 0.5-3

crosslinker molecules per antibody molecule. Next, the derivatised antibody

is separated from the crosslinker, using any number of standard biochemical

separation techniques. Preferably the separation procedure will be

accomplished using a gel filtration column, and more preferably Sephadex G-25

(Regd. T.M. Pharmacia Corp.) will be employed. The column is pre-

equilibrated with a chromatographically compatible aqueous buffered solution.

The isolated derivatised antibody can then be reacted with the oligonucleotide

having a sulphydryl group as described below.

Oligonucleotide having a free sulphydryl group can be directly reacted with the

derivatised antibody in an aqueous buffered solution compatible with the

reaction. The oligonucleotide and antibody concentrations, and the duration of

the reaction may vary depending on the number of oligonucleotide molecules

sought to be bound to antibody. The reaction is preferably run at 4°C

overnight. Synthesis of the Preferred Oligonucleotide-Antibodv Con^jugates

More specifically, the synthesis of oligonucleotide-antibody conjugate is carried

out as follows.

The monoclonal antibody is reacted with the heterobifunctional crosslinker,

mal-sac-spacer-glut-HNSA- as follows. 10 mg/ml of MAb is reacted with a

two-fold molar excess of the thioether crosslinker in 0.1 M sodium phosphate,

pH 8, for about 25 minutes at room temperature. The progress of the reaction

is followed by measuring the absorbance at 406 nm. At the end of 25 minutes

the absorbance may have increased to 0.57, and the derivatised antibody is

separated from the reaction mixture by gel filtration using a Sephadex G-25

column (2.5 x 17 cm) in 40 mM sodium phosphate buffer, pH 6, containing

200 mM NaCl. This material is reacted, as described below, with the

oligonucleotide having a reactive sulphydryl group.

The oligonucleotide with reactive sulphydryl group was combined with

derivatised antibody in a 1:2 molar ratio (antibody: oligonucleotide with

sulphydryl group). The solution is concentrated using an Amicon stirred ultra-

filtration device. The buffer employed is 40 mM sodium phosphate, pH 7.6,

containing 200 mM NaCl. The reaction is allowed to proceed overnight at

4°C, and the sample is then chromatographed over GF 250 gel filtration column (PBS pH 7.6). The fractions collected are run on 6.5 % SDS-PAGE

and conjugates should be observed having molecular weight greater than the

unconjugated antibody.

Alternatively, to maximise the conjugation, the oligonucleotide with reactive

sulphydryl group is combined with derivatised antibody in a 1 :25 molar ratio

(antibody: oligonucleotides with sulphydryl group). The oligonucleotide and

antibody concentrations may vary depending on the number of oligonucleotides

bound to the antibody. Additionally, after the reaction has proceeded overnight

at 4°C, the sample can be chromatographed over a Sepharose (Regd. T.M.) S-

300 column (2.2 x 80 cm) in 40 mM sodium phosphate, pH 6.5, containing

200 mM NaCl. This step removed any unreacted oligonucleotides with the

sulphydryl group.

Fractions containing either free antibody, if any, or oligonucleotide antibody

conjugate can be identified using a suitable analytical technique, such as sodium

dodecyl sulphate polyacrylamide gel electrophoresis. The oligonucleotide

antibody conjugates so isolated can, if desired, be concentrated by any suitable

technique known in the art followed by sterilisation. The latter is readily

achieved by passing the oligonucleotide antibody conjugate through an 0.2

micron filter. The above conjugates may be as efficacious as conjugates formed by disulphide

linkages, and the methods of use of both types of conjugates are similar.

EXAMPLE 3

Selective Killing of SKBR3 Cells by Radiolabelled c-erb-B2 Oligonucleotides

The following oligonucleotides were synthesised containing phosphorothioate

linkages:

(a) Antisense

5'-CAC AAGGCCGCCAGCTC-3 '

This sequence is complementary to the non-transcribed sequence at the 5' end

of the gene.

(b) Sense

5'-GAGCTGGCGGCCTTGTG-3 '

This sequence is complementary to the transcribed sequence at the 5' end of the

gene. (c) Random

5-CTGGC ACG ACGC AC ACC-3 '

This is a random sequence, but has the same base-composition as the antisense

oligonucleotide.

The oligonucleotides were radiolabelled with iodine- 125 using the Iodogen

method of Mieczyslaw A. et al (1988) Analytical Biochemistry 172, 356-359.

Typically, ¹²⁵I-labelled oligonucleotides had a specific activity of 0.7MBq

mg^"

SKBR3 cells in 9 cm culture plates were grown in Dulbecco's modified MEM

containing 10% foetal calf serum supplement. Subconfluent cells were

detached using trypsin/versene, and plated out at a density of 1 x 10⁴ cells per

microtitre well (0.5 ml) in 24 well plates. Oligonucleotides (antisense, sense

or random) were added at 50 μM or 5 μM. Cells were then grown at 37°C in

an incubator with an atmosphere of 10% C0₂ in air. After 3 days cells were

harvested using trypsin/versene, at which time cells were counted and their

viability assessed by the trypan blue exclusion test.

Figure 1 shows the cell count when unlabelled random, sense and antisense c-

erb-B2 oligonucleotides are used to inhibit SKBR3 cells. The control shows the

effect of the same treatment but with no oligonucleotide present. The antisense oligonucleotide is considerably more effective in reducing cell viability than any

of the controls.

Figure 2 shows that the radioactive antisense oligonucleotide (*Anti) is

considerably more potent than the unlabelled random and antisense

oligonucleotide or the labelled sense oligonucleotides (*Sense). In Figure 2 all

cells were >96% viable immediately following treatment. The concentrations

given are those of the oligonucleotides, and the results indicate that the ¹²⁵I-

labelled antisense oligonucleotide appears to have a toxic effect on the SKBR3

cells at concentrations l/10th that of the unlabelled antisense oligonucleotide.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Epenetos, Agamemnon A (ii) TITLE OF INVENTION: Therapeutic Compounds (iii) NUMBER OF SEQUENCES: 20

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Eric Potter Clarkson

(B) STREET: St Mary's Court, St Mary's Gate

(C) CITY: Nottingham

(D) STATE: Nottinghamshire

(E) COUNTRY: United Kingdom

(F) ZIP: NG1 1LE

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: GB 9123947.5

(B) FILING DATE: 12-NOV-1991

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Bassett, Richard S

(C) REFERENCE/DOCKET NUMBER: IMPF/P9956GB

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: +44(0)602 585800

(B) TELEFAX: +44(0)602 588122

(C) TELEX: 37540 Potter G

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GCTGAAGGGC TTTTGAACTC TGCTTA 26

(2) INFORMATION FOR SEQ ID NO:2: 30

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

GCTGAAGGGC TTCTTCCTTA TTGATG 26

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GCTGAAGGGC TTCTGCGTCT CCAT 24

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

CTGGTCTAAC CAGAGAGACC 20

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GCAAGCTTTA TTGAGGCTTA 20

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

CAGTCAGTCA GTCAGTCAGT 20

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

CTGCTAGAGA TT 12

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear 32

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

TGCTAGAGAT TTTCCACAC 19

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

TTCAAGTCCC TGTTCGGGCG CCAAA 25

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

GCGTACTCAC CAGTCGCCGC 20

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: 1inear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

CTGCTAGAGA TTAA 14

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

ACACCCAATT CTGAAAATGG 20

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

TCTCCCTCTT AGCTGGTCCT CTGC 24

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES 34

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

CATGCTTTCA GTGCTCATGG TGTCCTTTC 29

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

GATCAGGAAG GAGAAGAGGC TGAGGAACAA 30

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

CTCAGCTTGA GGGTTTGC 18

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGYt linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSEΪ YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: TTCGTCCTCC TCACAGGGC 19

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

CACAAGGCCG CCAGCTC 17

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

GAGCTGGCGG CCTTGTG 17

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

CTGGCACGAC GCACACC 17

Claims

1. A compound comprising a means to direct the compound to a nucleic acid within a cell and a radioactive moiety capable of destroying adjacent biological matter.

2. A compound according to Claim 1 wherein the means of direction comprises an antisense oligonucleotide.

3. A compound according to Claim 1 or 2 wherein the radioactive moiety comprises iodine-125, iodine-131, phosphorus-32, rhenium-186, rhenium-188 or yttrium-90.

4. A compound according to Claim 2 or 3 wherein the antisense oligonucleotide is complementary to DNA or RNA specifically forming part of a gene for the mutant ras protein, the mutant p53 protein, the BCR ABL protein or an HIV (human immunodeficiency virus) protein.

5. A compound according to any one of the preceding claims additionally comprising a targeting portion to target the compound to cells generally or to a desired cell type.

6. A compound according to Claim 5 wherein the targeting portion is a monoclonal antibody or part thereof specific for a tumour-cell-specific or virally-infected-cell-specific entity.

7. A compound according to Claim 6 wherein the entity is expressed on the surface of the cell.

8. A method of synthesising a compound according to any one of Claims 1 to 7 comprising conjugating a radioactive moiety capable of destroying adjacent biological material and a means to direct the compound to a nucleic acid.

9. A pharmaceutical composition comprising a compound according to any one of Claims 1 to 7 and a pharmaceutical carrier.

10. A method of treating a mammal having biological matter to be destroyed, the method comprising administering a compound according to any one of Claims 1 to 7.